In recent years, an increasing number of formulations of pharmacologically active proteins or peptides have been developed for practical use. In general, proteins or peptides are difficult to prepare as oral formulations due to their low stability in the gastrointestinal tract and low absorption from the intestinal tract membrane. For this reason, proteins or peptides are mostly used as injections in clinical practice. However, proteins or peptides usually have a short half-life in blood, and hence must be administered repeatedly at frequent intervals when used as drugs, thus imposing excessive burdens on patients. There is therefore a demand for practical sustained-release formulations of proteins or peptides, which exert their efficacy in as small amounts as possible and which permit reduced frequency of administration. Likewise, in the case of low-molecular-weight compounds when administered as drugs, there is also a high need for long-acting formulations intended to prolong drug efficacy.
Moreover, sustained-release formulations of proteins or peptides have a problem in that denaturation or aggregation of the proteins or peptides will occur during formulation process or in the formulations after in vivo administration. Means for preventing such denaturation or aggregation to avoid a reduction in the recovery rate are very beneficial in terms of increasing their bioavailability.
Attempts have been made to prepare sustained-release formulations based on a biodegradable polymer matrix such as polylactic acid-polyglycolic acid copolymer (PLGA), but such formulations are reported to cause protein denaturation and/or aggregation due to matrix hydrophobicity and/or as a result of manipulations required for formulation (e.g., emulsification, drying, acidification) (see Non-patent Documents 1 and 2). On the other hand, there are also reports of sustained-release formulations based on a hydrophilic hydrogel matrix with reduced risks of these problems, but no such formulations are yet available for practical application. In terms of safety, materials used for formulations should have non-antigenicity, non-mutagenicity, non-toxicity and biodegradability. Thus, no sustained-release formulation is now ready for practical use in all aspects, i.e., encapsulation efficiency and recovery rate of proteins or peptides, as well as safety.
Some recent reports have proposed the use of polysaccharides as matrixes for drug carriers. Among them, hyaluronic acid (HA), a biomaterial (polysaccharide) isolated from the vitreous body of bovine eyes in 1934 by K. Meyer, has been known as a major component of extracellular matrix for a long time. HA is a kind of glycosaminoglycan composed of disaccharide units in which D-glucuronic acid and N-acetylglucosamine are linked to one another via β(1→3) glycosidic linkages. There is no difference among species in the chemical and physical structure of HA and humans also have a metabolic system for HA; HA is therefore one of the safest medical biomaterials in terms of immunity and toxicity. Recent years have enabled microbial mass production of high-molecular-weight HA and also have allowed practical use of HA in the fields of therapeutic agents for degenerated cartilage, cosmetics, etc.
There are some reports of an attempt to sustainedly release a protein or peptide as a drug from a gel which is composed of chemically crosslinked HA, because HA has non-antigenicity, non-mutagenicity, non-toxicity and biodegradability and appears to be preferred in terms of safety. Techniques known for gelling HA by chemical crosslinking include the carbodiimide (CDI) method (see Patent Document 1), the divinylsulfone (DVS) method (see Patent Document 2), and the glycidyl ether (GE) method (see Patent Document 3). Another technique is also known, in which HA is modified to have hydrazide (HZ) groups as crosslinking functional groups and the resulting HA derivative (HA-HZ) is then crosslinked with a crosslinking agent (see Non-patent Document 3).
There are also reports of sustained-release formulations which encapsulate a protein or peptide within a HA gel by in situ crosslinking (see, e.g., Patent Document 4). In the process for such in situ crosslinked sustained-release formulations, it is desired to have a smaller influence on drugs. To minimize reactions in proteins and peptides, a method has been reported that uses crosslinking reaction by oxidation of mercapto groups under mild conditions to prepare a drug-encapsulating HA gel (see Patent Document 5), but this method still has room for improvement when applied to proteins or peptides containing cysteine residues. Also, another method has been reported to minimize reactions with proteins or peptides, in which polyethylene glycol (PEG) is used as a matrix and crosslinked through nucleophilic addition reaction of unsaturated functional groups (see Patent Document 6), but this method has problems in reaction selectivity and versatility, and also suffers from a problem in that fragments of non-biodegradable PEG remain in the body, as described above.
Protein or peptide denaturation induced by side reactions may be responsible not only for reduced biological activity, but also for antigenicity development. Thus, reactions used for gel crosslinking must be highly selective without affecting a protein or peptide to be encapsulated. However, there is not known any in situ crosslinking method that is sufficient to solve all of these problems, i.e., reaction selectivity, versatility and safety.
In contrast, when a protein or peptide is encapsulated into a pre-crosslinked gel, it is advantageous in that side reactions between drug and matrix during chemical crosslinking can be completely avoided. Moreover, it is possible to remove the excess of crosslinking agent in the absence of the drug by washing the gel after chemical crosslinking and/or to eliminate unreacted crosslinking functional groups by reaction with another reactive group. Thus, it is also advantageous in that problems of contamination arising from unreacted crosslinking functional groups and residual crosslinking agent can be avoided. However, such an approach results in low encapsulation efficiency due to problems arising from compatibility and/or electrostatic repulsion between HA and protein or peptide, and it also has a problem in that it fails to provide sustained-release properties.
Previous reports have shown that a polysaccharide which is obtained by introducing hydrophobic groups (e.g., groups including a cholesterol group, an alkyl group and the like) into a hydrophilic polysaccharide (the polysaccharide thus obtained is hereinafter also referred to as “hydrophobized polysaccharide” or “HP”) spontaneously associates in an aqueous solution to form a nano-size particulate (nanogel) having a hydrogel structure (see Non-patent Documents 4 and 5), that this nanogel serves as a host molecule which is complexed with a hydrophobic low-molecular-weight substance, a peptide, a protein or the like (see Non-patent Documents 4, 6 and 7), and that this nanogel serves as an artificial molecular chaperone which facilitates heat stabilization and/or refolding of proteins (see Non-patent Documents 8 and 9). Moreover, further reports have been issued for hybrid gels prepared from this nanogel by being modified to have polymerizable groups (e.g., methacryloyl groups) and then crosslinked by copolymerization with functional monomers (see Patent Document 8 and Non-patent Document 10).
Patent Document 1: International Publication No. WO94/02517
Patent Document 2: JP 61-138601 A
Patent Document 3: JP 5-140201 A
Patent Document 4: U.S. Pat. No. 5,827,937
Patent Document 5: International Publication No. WO2004/046200
Patent Document 6: International Publication No. WO2000/44808
Patent Document 7: International Publication No. WO2004/050712
Patent Document 8: JP 2005-298644 A
Patent Document 9: International Publication No. WO2006/028110
Patent Document 10: International Publication No. WO00/12564
Patent Document 11: European Publication No. 0842657
Patent Document 12: International Publication No. WO2005/054301
Non-patent Document 1: J. Pharm. Sci., vol. 88, pp. 166-173, 1999
Non-patent Document 2: J. Microencapsulation, vol. 15, pp. 699-713, 1998
Non-patent Document 3: J. Am. Chem. Soc., vol. 116, pp. 7515-7522, 1994
Non-patent Document 4: Macromolecules, vol. 26, pp. 3062-3068, 1993
Non-patent Document 5: Macromolecules, vol. 30, pp. 857-861, 1997
Non-patent Document 6: Macromolecules, vol. 27, pp. 7654-7659, 1994
Non-patent Document 7: J. Am. Chem. Soc., vol. 118, pp. 6110-6115, 1996
Non-patent Document 8: Bioconjugate Chem., vol. 10, pp. 321-324, 1999
Non-patent Document 9: FEBS Letters, vol. 533, pp. 271-276, 2003
Non-patent Document 10: Biomacromolecules, vol. 6, pp. 1829-1834, 2005
Non-patent Document 11: J. Biomedical Materials Research, vol. 47, pp. 152-169, 1999
Non-patent Document 12: J. Controlled Release, vol. 54, pp. 313-230, 1998